Oxygen (O₂) at high pressures acts as a neurotoxic agent leading to convulsions. The mechanism of this neurotoxicity is not known; however, oxygen free radicals and nitric oxide (NO) have been suggested as contributors. This study was designed to follow the formation of oxygen free radicals and NO in the rat brain under hyperbaric oxygen (HBO) conditions using in vivo microdialysis. Male Sprague-Dawley rats were exposed to 100% O₂ at a pressure of 3 atm absolute for 2 h. The formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a result of perfusing sodium salicylate was followed as an indicator for the formation of hydroxyl radicals. 2,3-DHBA levels in hippocampal and striatal dialysates of animals exposed to HBO conditions were not significantly different from controls. However, rats treated under the same conditions showed a six- and fourfold increase in nitrite/nitrate, break down products of NO decomposition, in hippocampal and striatal dialysates, respectively. This increase was completely blocked by the nitric oxide synthase (NOS) inhibitor l-nitroarginine methyl ester (l-NAME). Using neuronal NOS, we determined the NOS O₂ K _m to be 158 ± 28 (SD) mmHg, a value which suggests that production of NO by NOS would increase approximately four- to fivefold under hyperbaric O₂ conditions, closely matching the measured increase in vivo. The increase in NO levels may be partially responsible for some of the detrimental effects of HBO conditions.